Alpha-crystallins are a family of small heat shock proteins that play a central role in lens development and the maintenance of lens transparency. Evidence suggests that ?-crystallins prevent lens cataracts during aging, and that mutations in these proteins can lead to congenital cataracts. Previous work in our laboratory and in others shows that lens development, lens biochemistry and ?-crystallin function are well conserved between mammals and zebrafish, supporting the use of zebrafish as a model system for lens biology. The goal of this current proposal is to develop a fast and cost efficient in vivo system fr assessing the functional properties of native and variant ?-crystallins by leveraging recent tools for manipulating gene expression in the zebrafish. In Aim I, undergraduate research students will be trained in an innovative genome editing technique (called CRISPR/Cas) to disrupt the three zebrafish ?- crystallin genes and produce genetically modified fish populations lacking combinations of each protein. A variety of morphological, histological and proteomic techniques will be used to assess the resulting impact of disrupting the function of ?-crystallin genes. Changes in protein expression, aggregation and post-translational modification in each functional knockout fish population will be examined to identify the role of ?-crystallins in les development and the modulation of age-related changes that can lead to cataract. The ability of zebrafish to produce large numbers of externally developing transparent embryos provides a powerful tool for identifying potential effects of ?- crystallin loss from the earliest stages of ye development to old age (2 years). In Aim II, a zebrafish mutant strain with congenital cataracts will be used as an in vivo model system for assessing the ability of native and modified ?-crystallins to prevent disease-causing protein aggregation. Students will construct DNA plasmids to drive the expression of specific ?-crystallin genes and microinject them into single-celled zebrafish zygotes. Increases in ?-crystallin expression will be quantified by targeted mass spectrometry and the ability of each expressed ?- crystallin to prevent cataract will be assessed by Nomarski light microscopy and confocal microscopy. The in vivo zebrafish tools produced in this study will complement the use of mammalian models and in vitro techniques for analyzing the roles of ?-crystallin in the lens. This work is relevant to vision researchers interested in te etiology and prevention of cataract, a leading cause of human blindness worldwide.